Several methods use immunoassay techniques for detection and quantification of antigen in a specimen. Two types of immunoassay systems are currently used. In a homogeneous system, the assay is performed in a single phase. In a heterogeneous system, the immunoassay is performed in two steps. The additional step is needed to separate bound material from unbound. Typically, a solid support surface is used as a bound phase, to which antibody or antigen is attached via adsorption or chemical bonding. Many kinds of solid support surfaces have been developed to improve the performance of an immunological reaction and the efficacy of the separation step.
Particles have been used as a mobile solid phase in agglutination assays to improve the efficiency of the immunological reaction between antibody and antigen. In said system, soluble antigens/antibodies will combine with specific antibodies/antigens bound onto the particles resulting in precipitates of insoluble antigen-antibody immunocomplexes on the surface of the particles. In solid phase immunoassays, the sensitivity of the assay can be increased by washing the solid phase bound immunocomplex, which results in more complete separation of unbound material and thus in increase of signal to noise ratio. Additionally, microparticles of various compositions have also been used in an effort to increase the surface area of the solid support.
Particle based agglutination assays are well-known for those skilled in the art. The original agglutination assays exploited single sized particles as polystyrene particles i.e. latex particles. A method to overcome the low sensitivity of the agglutination assay especially when assaying small quantities of an analyte is disclosed in U.S. Pat. No. 4,279,617, where two different coated particulate reagents are used. Said particles may be of different size. Said method does not involve a separation of unbound material from the agglutinate using a filtration process.
Prior art documents acknowledge a number of publications disclosing assay techniques which rely on the use of a porous member such as a membrane, filter or other matrices. U.S. Pat. No. 4,632,901, EP-B-180638 and U.S. Pat. No. 4,727,019 disclose a member to which is bound a receptor for the target analyte and a second member to draw liquid added to the first member. When a liquid sample is added, the liquid flows through and the receptors in the member bind the analyte present in the sample. After the addition of the sample, another receptor for the analyte which is labelled is added to permit detection. Suitable labels being an enzyme, radionuclide or fluorescent label. When an enzyme label is used a disadvantage is that a colouring forming substrate must be added to the membrane.
EP-B-253579 discloses a further embodiment of the above mentioned member-based technique. The embodiment takes advantage of microspheres which are entrapped within the interstices of the porous membrane in order to embrace a means for fixing receptor or anti-receptor to the porous member.
U.S. Pat. No. 4,916,056, EP-B-389003, U.S. Pat. No. 5,008,080 and U.S. Pat. No. 5,149,622 disclose membrane based flow through assays exploiting particles onto which antibodies or antigens have been retentioned. The size of the particles is not critical although it is preferred that the size is smaller than the average pore size of the fibrous matrix.
Three dimensional membranes are commonly used for separation of immunocomplex from unbound material. U.S. Pat. No. 5,501,949 discloses an immunoassay using finely divided particles as a solid phase for the first binding component (substance). The soluble, second binding components are labelled with a signal generating material. The soluble, non-particle bound, labelled binding component used in this method has lower specific activity compared to binding component labelled with a particle bound label. This results in the loss of sensitivity of the assay. The method uses a three dimensional membrane for filtering the immunocomplex and separating the unbound material.
U.S. Pat. No. 4,853,335 discloses an immunoassay where a biological specimen, colloidal gold labelled ligand or anti-ligand, and solid phase capture particles coated with ligand or anti-ligand are applied on a porous film. The particles captured on the membrane are visually inspected for colour.
GB Application No. 2123146 describes an assay method accomplished in a dual channel optical-electrical cell counter or in a fluorescence microscope. The disclosure involves first and second microscopic particles having different detectable properties by performing said assay methods. Said application does not disclose any separation of unbound material from agglutinate using a filtration process. A biological fluid is intermixed with particles, whereafter their different properties are measured.
U.S. Pat. No. 5,565,366 comprises a method where a test mixture is formed by contacting a sample with coloured particles which bear on their surface receptors specific for the ligand. When the test mixture is passed through a filter having apertures larger than the particles but smaller than the aggregates, the aggregates are removed from the filtrate and the colour of the filter/filtrate is analysed. The document discloses that the used small particle size is more advantageous with regard to aggregate formation than larger particles used in prior art techniques. However, the use of small particles is disadvantageous since the consumption of ligand or anti-ligand on the particle is higher the smaller the particles are.
U.S. Pat. No. 6,268,222 discloses a different approach where microparticles are attached to nanoparticles labelled with fluorescence dye. The particles are attached to each other by their surface functional groups. This invention comprises a core or carrier particle having on its surface a plurality of smaller polymeric particles. When different fluorescence dyes are used, multiple fluorescence emission is obtained by a light source excitation. By varying the quantity and ratio of different populations of nanospheres, it is possible to establish and distinguish a large number of discrete populations of carrier particles with unique emission spectra. This approach is useful for multiplex analysis of a plurality of analytes in a sample.
As disclosed in the above review of prior art, particles alone or together with different applications of three dimensional membranes have been used to separate desired analytes from a sample. However, there is often unspecific binding of labelled binding substance due to the porosity of the membrane. Thus, the signal to noise ratio is low, due to a high background signal, when using three dimensional membranes. Hence, three dimensional membranes require considerable washing to reduce unspecific binding.
In addition to the methods disclosed above, assays exploiting, for example, magnetic beads are also well known for those skilled in the art.
The methods disclosed in the above prior art documents rely mainly on visual detection of analyte and are, therefore, not sensitive enough to measure low concentrations of analyte. Moreover, the level of signal created by the label is not, in all circumstances, intensive enough.
More sophisticated methods in the art apply the change in electrical current to indicate presence of analyte in sample. Field effect transistors coated with a layer of antibody in the gate region are utilised, for example, in U.S. Pat. No. 4,238,757 to detect antigen-antibody reaction by a change of charge concentration of the transition.
A tagged reagent which reacts with the analyte-reagent complex or with the reagent to change the electrical reactance of the surface is added in U.S. Pat. No. 4,219,335.
An immunologic reaction can also be measured by a voltameric immunoassay as disclosed in U.S. Pat. No. 4,233,144, where one immunoreactant is labelled with an electroactive substance.
U.S. Pat. No. 4,054,646 discloses a method where an antigen-antibody layer is sandwiched between two conducting layers and where the electric capacitance of the resulting laminate is measured.
Another type of capacitance-measuring technique which exploits a pair of electrodes coated with a substrate and immersed in a medium containing a material which specifically binds with the substrates is disclosed in U.S. Pat. No. 4,072,576.
It is also possible to combine a change effect signal detection with an enzyme immunoassay technique as described in U.S. Pat. No. 4,287,300.
However, the above electrical methods did not meet the needs of a simple, fast, sensitive, inexpensive and easy-to-use method to perform an immunodiagnostic assay.
A method intended to solve the above mentioned disadvantages is disclosed in U.S. Pat. No. 5,284,748 where antigen or antibody-labelled colloidal gold particles are employed optionally with silver enhancement in a new immunodiagnostic method. The complex so formed causes full or partial completion (closing) of an essentially open electrical circuit. A further embodiment comprises a pair of spaced-apart electrical conductors, particularly conductive layers disposed on a substantially non-electrically conductive base. There is a space between the conductors defined as a path or channel. Means forming an electrical circuit is contacted to each of the conductors so that the channel constitutes a break in the circuit. The binding reaction between the pair of substances is responsible for fully or partly bridging the break in the circuit. One such means involves adhering one of the substances to the surfaces of electrically conductive particles.